We have characterized a novel class of thymidine kinase (tk) negative mutants of Herpes simplex virus which lack a full-size tk polypeptide. Conclusive identification of the tk polypeptide and the relatedness of novel short polypeptides produced by these mutants was accomplished by partial purification, peptide analysis, and positive mRNA hybridization-translation. DNA from pBR322 containing a tk gene insert was used as a hybridization probe in conjunction with "Southern and Northern" blotting techniques to analyze the tk gene and mRNA structure. Our results are consistent with the possibility that some of the tk minus mutants arise through premature termination of polypeptide synthesis due to nonsense or frameshift mutation. We are presently trying to identify some of these mutants as nonsense mutations by in vitro synthesis and suppression with yeast suppressor tRNAs. These mutants are also being used to analyze viral tk gene expression in tk plus DNA transformed Ltk minus cells.